Results

- Cytological Aspects of the Differentiation of Barb Cells During the Formation of the Ramus of Feathers

Histology of barb ridges. Unless specifically indicated the morfog鮥sis of feathers was similar in the two species. Feather filaments were surrounded by a multilayered sheath at stages 36-38 in the chick or 13-14 days post-deposition in the zebrafinch (Fig. 1 A). A similar histogenetic process was also noted in juvenile feathers of zebrafinch at 1 week post-hatching. Within these filaments long columns of cells were seen in longitudinal section which appeared as barb ridges in cross-section (Fig. 1A-C). A flat epithelium formed the

marginal plates bordering barb ridges (Fig. 1C, see ultrastructure later). The detailed examination of longitudinal sections of feather filaments showed the beginning of differentiation of barbs and barbules (Fig. ID). While barb medullary cells formed by piling up of cylindrical cells, barb cortical cells were seen as parallel fusiform cells forming a wall around medullary barb cells. More externally, fusiform barbule cells formed syncitial tubes reaching underneath the sheath. The innermost part of the sheath was made of pale, barb ridge vane cells. The central part of the feather filament contains mesenchymal cells and blood vessels.

At HH stages 39-40 in the chick large part of the feather filament (apical to more basal portions) was made of centrally located, keratinized barbs and by peripheral barbules (Figs. IE andF). In longitudinal sections, barb cells resemble tree-branches, with the thin barbules branching at regular intervals along the barb (ramus, Fig. IE). The branching of proximal (basal) barbule cells occured at opposite, right and left, sides on the barb. Barbules were made of several cells (5 or more) piled up to form narrow chains of barbule cells (see schematic drawing in Fig. 7). Barb medullary cells formed pale vacuolated centers surrounded by denserbarb cortical cells, the latter in continuity with barbule cells (Fig. 1 E, F). At late stages of feather morphogenesis (stage 40 in the chick and at 15-16 days post-deposition in zebrafinch) marginal plate cells and supportive cells present among barbule cells were largely disappeared. Large blood vessels were either degenerating or contained few blood cells.

Ultrastructure. An outer periderm and 3-5 layers of sheath cells were seen enwrapping the feather filaments at HH stages 37-38 in the chick and at 12-13 days in zebrafinch or in 1 week hatchlings. Pale barb ridge vane cells were interposed between the sheath externally and cells of barb ridges internally (Fig. 2). Cross sections of feather filaments in both species showed that barb ridges were made of a peripheral layer of cylindrical cells (presumptive marginal plates) and an inner axial plate made of irregularly aggregated cells (presumptive barb cells). The latter were in continuity with two external areas of more or less piled cells (alar plates, the presumptive barbule cells) separated by a central region of smaller cells (presumptive axial plate). Barb ridges were in contact with the external 4-5 cell-layered sheath by the interposition of paler cells with circular orientation, the barb vane ridge cells (Fig. 2).

At this stage of cytodifferentiation most cells of the barb ridge contained evenly distributed ribosomes, no endoplasmic reticulum and the nucleus was relatively euchromatic. However, barb and barbule cells often contained glycogen granules evenly distributed or clumped in variably-sized massess (Fig. 3). Vesicles of small size (0.1-0.4 |jm) were more or less evenly seen in the cytoplasm. The vesicles appeared to derive from the enlargment of the endoplasmic reticulum or from swelling and disappearing of cristae in mitochondria.

At later stages, 38-39 in the chick, 14-15 days and 1 week post-hatching in the zebrafinch, the vesicles in barb medullary cells increased and tended to fuse into large vesicles (Fig. 4). These vesicles appeared to contain a lipid-like material with very low density and their initial membrane appeared partially or largely interrupted (Fig. 3 B). Large lipid vesicles were more relevant in barb medullary cells of the zebrafinch, especially in feathers of 1 week post-hatchlings.

In the chick at HH stages 39-40 some amorphous material was still present within the large vesicles while keratin bundles accumulated along the cell periphery and joined the plasma membrane (Fig. 5 A). While the plasma membrane among barb cells showed small area of fusion or tight junctions at HH stages 37-38, large part of the membrane appered lost in the following stages, especially in rami of apical areas of feather filaments. At HH stages 39-40 some modified junctions were seen among barb cells, and they were composed by two dense peripheral lines and a thicker dense midline (Fig. 5 C). In the zebrafinch at 15-16 days post-deposition or in rami of feathers of 1 week post-hatchingls, most barb medullary cells were filled with lipids or partially empty (Fig. 6). While barb cortical cells were filled with keratin bundles, only the peripheral cytoplasm of barb medullary cells was keratinized forming trabeculae containing some lipids, occaional granular material (perhaps glycogen remnants), or empty cavities (Fig. 6). The lipidization of barb medullary cells was contemporary to that of interbarbule supportive cells at late stages of differentiation so that barbules became eventually independed and joined to the medullated ramus as indicated in Fig. 7.